In a surprisingly large number of mouse gene knockout studies, a common phenotype has been observed which involves a failure of fetal ventricular cardiomyocytes to proliferate during development and form a thickened outer ventricular chamber wall (the "compact zone"). These genes are the retinoic acid receptor (and nuclear transcription factor) RXRalpha, the cell cycle regulator N-myc, the transcription factors TEF1 and WT1, the cell surface receptor GP130, the cell surface receptor kinase (betaARK1, and the cell adhesion molecules VCAM1 and alpha4 integrin. Little is known of the manner in which most of the above-mentioned genes act. The assignment of these genes which regulate compact zone cardiomyocyte proliferation to their tissue of action and to pathways of action will define the nature of the events which underlie cardiac ventricular morphogenesis. Specific Aim 1: Define the cell autonomy of the TEF1, WT1, GP13O, and betaARK1 mutant phenotypes. New data indicates that RXRalpha functions outside of the cardiomyocyte lineage, and other investigators have determined the site of action of N-myc, alpha4 integrin, and VCAM1. By derivation of embryonic stem cells lacking TEF1, WT1, GP130, or betaARK1, and by evaluation of the behavior of these cells in chimeric embryos, it will be possible to determine if these genes act within or outside the cardiomyocyte lineage. Specific Aim 2: Evaluate candidate tissues as the site of action of RXRalpha. A combination of transgenic and chimeric approaches will define the tissue in which RXRalpha function is required for normal cardiomyocyte proliferation in the ventricular chamber wall. Specific Aim 3: Evaluate candidate tissues as the site of action of other genes which act in a cell nonautonomous manner. Transgenic and chimeric approaches will be employed to address the site of action of TEF1, WT1, GP130, and betaARK1 (if these act outside of the cardiomyocyte lineage), and to define pathways which unite the deficiency of these gene products into a common mutant phenotype. A greater understanding of the manner in which the genes described in this proposal act might shed light not only on the etiology of certain congenital heart defects, but also on general mechanisms which regulate the distinction between atrial and ventricular morphogenesis (the atrial chamber not undergoing formation of a compact zone), and on the loss of proliferative competence in postnatal cardiomyocytes.